Dengue viruses, members of Flaviviridae, are recognized as the causative agent of mosquito borne viral diseases affecting about 100 million people annually. Of these, more than 250,000 cases involve life threatening dengue hemorrhagic fever / dengue shock syndrome. The long term goal of this laboratory has been to dissect the biochemical mechanisms involved in dengue viral replication in the host. The specific objectives of this proposal are to define the specific molecular events during viral replication. An in vitro template dependent viral RNA replicase assay and the dengue infectious clone for in vitro analysis will be used to achieve these objectives, by fulfilling the following specific aims: 1. Conserved regions of the viral RNA required for both minus and plus strand RNA synthesis will be analyzed using the wild type and mutant subgenomic RNA templates in the viral replication assays. The subgenomic RNAs will be analyzed using RNA probing methods. The PI will analyze the effect of specific mutations on RNA synthesis using the viral replicase in vitro, and the growth phenotypes and replication efficiencies in vivo using the dengue infectious clone. 2. Viral replicase complexes will be isolated from recombinant vaccinia viruses expressing the wild type and mutant dengue viral protein precursor, which undergo processing and assembly in the infected cell in vivo. To study the mechanism of replication in the absence of virion assembly, a subgenomic dengue virus replicon expressing a readily quantifiable reporter gene product will be constructed. 3. Two hypotheses will be tested, using site directed mutants of NS3 in the in vitro viral replicase assay and the in vivo assay of mutant genomic RNAs trans-complemented by wild type NS3. First, the role of NS3, having NTPase and RNA helicase activities, is required for RNA synthesis at the 3' end in vitro. Second, a novel motif of NS3, Leu-Lys-Pro-Arg, is required for the 5'RNA triphosphatase activity of NS3. This enzyme activity is required in the first step for 5' capping of many viral RNAs. The PI propose that 5' capping is required for replication of RNA. 4. NS3/NS5 interaction is regulated by phosphorylation and subcellular location of NS5. The PI proposes to test the hypothesis that the nuclear NS5 regulates the expression of cellular gene(s) in response to viral infection.